Effects of ethanol on GluN1/GluN2A and GluN1/GluN2B NMDA receptor-ion channel gating kinetics.
Effects of ethanol on GluN1/GluN2A and GluN1/GluN2B NMDA receptor-ion channel gating kinetics.
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DOI：
10.1111/acer.14965
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发表时间：
2022-12
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--
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N-甲基-D-天冬氨酸受体(NMDAR)是酒精在中枢神经系统中作用的主要分子靶点，但酒精调节该离子通道活性的许多方面仍不清楚。我们和其他人已经证明，乙醇抑制NMDAR涉及门控的改变，特别是平均开放时间的减少。然而，关于乙醇对NMDAR动力学影响的完整描述，包括对动力学模型的拟合，尚未见报道。为了确定乙醇对NMDAR动力学的影响，我们在转染重组GluN1/GluN2A或GluN1/GluN2B NMDAR亚基的HEK-293细胞的外向外斑片上使用稳态单通道记录。使用非常低的谷氨酸浓度来分离该受体的单独激活。在两种亚基类型中，乙醇在接近全细胞IC50值(156 mM，GluN2A；150 mM，GluN2B)时，开放概率(Po)降低约50%，平均开放时间减少，开放频率不变。打开和关闭时间分布分别表现为两个和五个分量；乙醇选择性地降低了较长打开时间分量的时间常数和相对比例。在GluN2A亚基中，乙醇增加了除最长关闭时间外的所有组分的时间常数，而在GluN2B亚基中，乙醇不改变关闭时间。对动力学模型的门控部分的开放(代表受体的单独激活)的拟合表明，乙醇改变了两个速率：与GluN2A或GluN2B亚单位激活相关的速率，以及与关闭两个开放状态中较长的一个相关的速率。这些结果表明，乙醇选择性地改变个体的动力学速率，因此似乎选择性地影响NMDAR门控中涉及的不同构象转变。
The N-methyl-D-aspartate receptor (NMDAR) is a major molecular target of alcohol action in the central nervous system, yet many aspects of the manner in which alcohol modulates the activity of this ion channel remain unclear. We and others have shown that ethanol inhibition of NMDAR involves alterations in gating, especially reduction of mean open time. A full description of ethanol’s effects on NMDAR kinetics, however, including fitting to a kinetic model, has not been reported. To determine ethanol’s effects on NMDAR kinetics, we used steady-state single-channel recording in outside-out patches from HEK-293 cells transfected with recombinant GluN1/GluN2A or GluN1/GluN2B NMDAR subunits. Very low glutamate concentrations were used in order to isolate individual activations of the receptor. In both subunit types, ethanol, at approximate whole-cell IC50 values (156 mM, GluN2A; 150 mM, GluN2B), reduced open probability (Po) by approximately 50%, and decreased mean open time without changing frequency of opening. Open and shut time distributions exhibited two and five components, respectively; ethanol selectively decreased the time constant and relative proportion of the longer open time component. In the GluN2A subunit, ethanol increased the time constants of all but the longest shut time components, whereas in the GluN2B subunit, shut times were unchanged by ethanol. Fitting of bursts of openings (representing individual activations of the receptor) to the gating portion of a kinetic model revealed that ethanol altered two rates: the rate associated with activation of the GluN2A or GluN2B subunit, and the rate associated with closing of the longer of the two open states. These results demonstrate that ethanol selectively alters individual kinetic rates, and thus appears to selectively affect distinct conformational transitions involved in NMDAR gating.
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